Molecular Basis of Potassium Channels in the Kidney

描述(由申请人提供)：本提案的主要目标是从机制上解释鲜为人知的转运过程，这些过程驱动基底膜定位和生理调节远端肾单位两个密切相关通道(Kir2.3和Kir4.1，突变与East/Sesame综合征相关)的细胞表面密度。该计划在逻辑上建立在我们最近的发现基础上，定义了这些渠道中的贩运信号，并阐明了与它们相互作用的细胞内分拣和保留机制。具体地说，我们将解决以下关键和及时的问题：1.KIR渠道中的高尔基出口补丁如何影响基线分类？与通常由短线性多肽序列组成的传统运输信号不同，我们发现嵌入其三级结构的残基决定了典型的KIR通道的高尔基退出。这个信号补丁形成一个识别位点，与AP1a适配器复合体相互作用，从而标记通道，以便在反式高尔基体上整合到笼蛋白包裹的小泡中。在这里，我们测试了在Kir2.3和Kir4.1中发现的保守的斑块信号通过选择通道作为货物进入笼蛋白包裹的小泡而启动极化运输的假设。2.如何解释KIR航道C-终端区域的基侧贩运信号？基于我们已发表的和初步的观察，我们认为一旦通道被标记为包含在笼蛋白包裹的小泡中，其他信号就会通过相互作用的基侧运输伴侣蛋白(S)直接基侧输送。3.钾适应过程中肾皮质集合管基底外侧KIR通道重构的基础是什么？这一目的是为了验证一种假说，即先前未知的KIR通道重塑过程，包括Kir2.3和Kir4.1以及基侧PDZ保留复合体，支持了钾适应中基侧膜电导的增加。通过解决这些问题，调查计划将为控制健康中钾分泌的基本贩运机制提供新的见解，并了解当贩运信号和贩运机制在疾病中出现问题时会发生什么。 与公众健康相关：维持钾平衡的钾通道必须精确地组织在肾脏的两个极化膜域上，以便有效地分泌肾脏的钾。事实上，离子通道运输和表面表达的中断可能会对盐分和矿物质平衡造成毁灭性的后果。尽管它很重要，但在细胞生物学和生理学中，一个长期存在的基本问题是如何精确控制这些膜蛋白的数量和位置。在目前的提案中，我们阐明了在健康中驱动这些通道膜运输的分子机制，并研究了当这些过程在疾病中出错时可能发生的情况。因此，这些研究将为肾脏K处理和K动态平衡在健康和疾病中的分子基础提供新的见解，同时阐明肾脏中膜蛋白靶向的基本机制。

DESCRIPTION (provided by applicant): The overarching goal of the present proposal is to develop a mechanistic explanation of the poorly understood trafficking processes that drive basolateral membrane localization and physiologically regulate the cell surface density of two closely related channels (Kir2.3 and Kir4.1, mutations in which are associated with EAST/Sesame syndrome) in the distal nephron. The program logically builds on our recent discoveries, defining the trafficking signals in these channels and the elucidating the intracellulr sorting and retention machinery that interact with them. Specifically, we will address the following critical and timely questions: 1. How does the Golgi Export patch in Kir channels influence basolateral sorting? Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, we discovered that residues embedded its tertiary structure dictate Golgi exit of a prototypical potassium Kir channel. This signal patch forms a recognition site for interaction with the AP1A adaptor complex, thereby marking channels for incorporation into clathrin-coated vesicles at the trans- Golgi. Here we test the hypothesis that the conserved patch signal found in Kir2.3 and Kir4.1 initiates polarized trafficking by selecting channels as cargo for inclusion into clathrin-coated vesicles. 2. How are the basolateral trafficking signals in the C- terminal region of Kir channels interpreted? Based on our published and preliminary observation, we propose that once channels are marked for inclusion into clathrin-coated vesicles, other signals direct basolateral delivery by interacting basolateral trafficking chaperone(s). 3. What is the basis for basolateral Kir channel remodeling in the renal cortical collecting duct during potassium adaptation? This aim is designed to test the hypothesis that a previously unrecognized Kir channel remodeling process, involving Kir2.3 and Kir4.1 and a basolateral PDZ retention complex, underpins the increase in the basolateral membrane conductance in potassium adaptation. By addressing these questions, the program of investigation will provide new insights into the fundamental trafficking mechanisms that control potassium secretion in health and to understand what happens when trafficking signals and trafficking machiery goes wrong in disease. PUBLIC HEALTH RELEVANCE: Potassium channels that underpin potassium balance must be precisely organized at two polarized membrane domains in the Kidney for efficient renal potassium secretion. Disruption of ion channel trafficking and surface expression can, in fact, have devastating consequences on salt and mineral balance. Despite its importance, a long-standing and fundamental question in cell biology and physiology has been how the number and location of these membrane proteins are precisely controlled. In the present proposal, we elucidate the molecular mechanisms driving membrane trafficking of these channels in health and study what may happen when these processes go awry in disease. Thus, the studies should provide novel insights into the molecular basis of renal K handling and K homeostasis in health and disease while illuminating fundamental mechanisms of membrane protein targeting in the kidney.